Bt Research 2012, Vol.3, No.3, 11
-
19
18
immersion in a solution of osmium tetroxide (OsO
4
)
at 2% in sodium cacodylate buffer for 1 h. The
samples post-fixed in OsO
4
were washed twice in
cacodylate buffer 0.1 M, at a 10 min interval, and
twice in distilled water at a 5 min interval; soon after
this, they were submitted to dehydration in a graded
ethanol series (30%, 50%, 70%, 90% and 100%),
being left for 10 minutes in each of the concentrations.
The samples were washed three times for 20 min each
time in ethanol at 100%. After this, the samples were
dried by the CO
2
critical point method, in a Baltec
CPD 030 appliance, and after being mounted on a
metal support were sputter-coated with about 25 nm of
gold using a MED 010 appliance from Balzers. The
samples that had been thus prepared and duly
identified were observed in a scanning electron
microscope from Zeiss, model DSM 962.
3.7
Detection of
B. thuringiensis
marked with
methionine
35
S on cabbage
Cabbage seeds were disinfected, as in item 4, and
sown in a centrifuge tube of 50 mL, containing 15 mL of
MS medium; they were then kept for 15 days in an
acclimated room. After 13 days, three bacterial
strains were inoculated separately in Petri dishes
containing Embrapa Agar with methionine
35
S added
at a concentration of 10
Ci/
L, and were kept at
room temperature for 48 hours. Next, the dishes were
scraped with a Pasteur pipette and the bacterial mass
of each of the strains was mixed with 5 mL of PBS
buffer. Of the 5 mL, 1 mL of each strain was applied
to one of the seedlings. One of the seedlings was left
as a control. Five days later, the seedlings were
placed inside cellophane paper to dry in a gel drier
for one hour. Then the seedlings were conditioned in
a lead cassette and, in the dark, were exposed to an
auto-radiograph film for a period of 30 days. After
this, the film was revealed for observation of
radio-marking.
Authors’ Contribution
LP conducted all the research for this paper and prepared the
manuscript. AG participated in seedlings preparation for
microscopic analyses. GC participated in the experiments of
plant growth promoter. ESM participated in activities with
radioactivity. ES participated in data analysis and reviewed the
manuscript. RM coordinated the project and was involved in
manuscript preparation.
Acknowledgement
This work was financially supported by FAP-DF and Embrapa.
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